Method of treating biological tissue by microwave-irradiation

ABSTRACT

A method of efficiently removing or fixing donor cells from a native tissue of mammalian origin comprises immersing the tissue in a treating solution, and irradiating the tissue with microwave while maintaining the temperature thereof in the range between 0° C. and 40° C.

FIELD OF THE INVENTION

The present invention is in the field of regenerative medical technologyin which the function of a particular organ or tissue of a patient isnormalized by transplantation when the function is lost or otherwiseabnormal. More particularly the invention relates to a method forpreparing transplantable tissues from native tissues of mammalian originby removing cell components or fixing the tissue using a fixing agentsuch as glutaraldehyde.

BACKGROUND ART

Scafold materials are prepared from native tissues for clinicalapplication by chemically treating the tissue with a fixing agent suchas glutaraldehyde or by decellularizing the tissue. In heart valvereplacement, for example, xenogeneic heart valves are prepared fromporcine heart valves or bovine pericardia by treatment withglutaraldehyde to diminish their immunogenicity. These xenogeneic valvesare highly anti-clotting but durable only for 5-10 years in youngrecipients. Therefore, they are normally transplanted to olderrecipients over 60 years old.

Since tissue bank systems have been organized in Europe and Americaaround 1985 and also in Japan in recent years, allogeneic cryopreservedvalves from deceased donors have been clinically used. The allogeneicvalves are less thrombogenic than mechanical valves, more durable thanxenogeneic valves and less susceptible to infections than both. However,a critical problem is the fact that the number of available valves isabsolutely insufficient. Moreover, cases in which functional failureappeared at a relatively early stage have been reported among youngrecipients suggesting the involvement of immune reactions. In the Rossoperation know to be effective in young recipients, autologous pulmonaryvalve is transplanted to aortic valve site and the impaired pulmonaryvalve is reconstructed with cryopreserved allogeneic valve. Thecharacteristic feature of the autologous pulmonary valve transplanted tothe aortic valve site is that it is growable as the recipient grows. Incontrast, mechanical valves and xenogeneic valves as well ascryopreserved allogeneic valves cannot grow and re-transplatation isoften needed for children. In order to eliminate the above problems,several studies have been reported removing donor cells from allogeneicvalves so that their immunogenicity and involvement of immune reactionsare diminished to increase the durability and autogenesis oftransplanted valves.

A decellularization method using a chemical solution called “SynerGraft”was developed by CryoLife, U.S.A. It was reported that thedecellularized tissue by this method was infiltrated into autologouscellular structures within several months and recellularized withautologous cells.

Harverich et al. of Hannover University, School of Medicine, Germanypublished a decellularization method using the detergent Triton X-100and proteolytic enzyme tripsin solutions.

However, washing with detergent or other chemical solutions alone is notsufficiently effective to remove bacteria, viruses and othercontaminants from the interior of tissue because the washing depends ondiffusion and penetration of the washing solution from surfaces of thetissue. Because of these limitations, complete decellularization andremoval of bacteria and viruses are hardly possible for large tissuematerials. In order to achieve satisfactory effects by chemical washing,it is necessary to increase the degree of treatment. This may lead toproblems of post-graft calcification and removal of residual treatingchemicals. As evidenced from BSE and CJD infections in transplantation,safety assurance is very important for the tissue to be transplanted.Currently known treating processes do not assure complete inactivationof viral contaminants and infection incidents may often occur fromtransplanted tissue contaminated with viruses.

The decellularized xenogeneic or allogeneic tissues are recellularizedby seeding and culturing autologous cells for transplantation as ahybrid regenerative tissue.

DISCLOSURE OF THE INVENTION

It is, therefore, an object of this invention to provide a method whichcan eliminate or ameliorate the disadvantages of the prior art, namely,a method which can accomplish, first, removal of cellular components,bacteria and viruses from large size tissues, second, can enabletreatment without impairing the biomechanical properties of the tissue,and, thord, allows sterilization of the tissue in a simple manner in ashort period of time.

The present invention, provides a method of treating native tissues ofmammalian origin comprising immersing said tissue in a treatingsolution, and irradiating said tissue with microwaves while maintainingthe temperature thereof at a temperature in the range between 0° C. and40° C.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1 is a schematic illustration of an exemplary system for carryingout the present invention.

FIG. 2 is a microscopic view of a specimen of porcine heart valve tissuetaken in cross-section. The specimen treated with the prior art solutionis shown on the left while the specimen treated with the prior artsolution in conjunction with microwave-irradiation according to thepresent invention is shown on the right. Residual nuclei are observed inthe interior of the tissue treated with the prior art solution alone(lower left).

FIG. 3 is a graph showing the efficiency of removal of Triton X-100 fromdecellularized porcine heart valve. The method of the present inventionenables the time required for removal of Triton X-100 to be decreased toabout 1/10 compared to the prior method.

BEST MODE FOR CARRYING OUT THE INVENTION

The method of the present invention involves treatment of native tissuesto prepare transplantable tissues by decellularizing of the nativetissues. In this case, the treating solution may be pure water, ahypertonic solution, a hypotonic solution, a detergent solution, anenzyme solution, a liquid medium, or a mixture thereof with a smallproportion of an organic solvent.

The method of the present invention also involves preparingtransplantable tissues by fixing native tissues. The treating solutionin this case is a solution of fixing chemicals such as glutaraldehyde.

In either treatment, the prior art method without microwave-irradiationrequires a relatively long period of time until the treating solutionmigrates throughout the tissue because the solution gradually diffusesand penetrates from the tissue surfaces. This may result in a risk ofcontamination of the tissue. The irradiation of native tissues withmicrowave in accordance with the present invention enables the treatingtime required for penetrating the treating solution throughout thetissue to be decreased to about 1/10 compared with the prior art method.Thus substantial improvement in the treatment efficiency may be achievedwhile preventing the tissue from contamination. Furthermore, the presentinvention can decellularize the tissue even from deep portions in ashort period of time which has been otherwise difficult or impossible toaccomplish.

Microwaves have hitherto been used in the field of histopathology e.g.for tissue fixing, bone decalcification, or defatting purposes, and inhistoimmunological chemistry. It is believed, however, that theapplication of microwave for the purpose of preparation oftransplantable tissues was not known.

In the new method according to the present invention, the native tissueof mammalian origin is placed in a container made of amicrowave-transmitting material such as glass or a plastic. Then atreating solution is poured into the container until the tissue iscompletely immersed in the solution. The treating solution may be adetergent solution, a hypotonic solution or a hypertonic solution whendecellularization is intended, or a solution of fixing chemicals such asglutaraldehyde when cell fixing is intended. The tissue in the treatingsolution is then irradiated with microwave while maintaining the tissueat a temperature between 0° C. and 40° C. Because the tissue is heated,it is necessary to cool the tissue during the irradiation withmicrowave. This may be accomplished using a commercially available rapidmicrowave-treatment apparatus by providing the apparatus with coolingmeans. To this end, the container containing the tissue and the treatingsolution is place in the microwave oven and a antifreezing coolant iscirculated between the container and a cooling apparatus external of themicrowave oven. A temperature sensor is disposed in the tissue containerto control the microwave oven and the cooling apparatus in response tothe sensed temperature to maintain the tissue at a temperature between0° C. and 40° C. Apart from histopathological procedures, preparation oftransplantable tissues requires preventing denaturing of tissue matrixso as to preserve the biomechanical properties thereof. Therefore, it isimperative to avoid temperature below 0° C. or above 40° C. duringirradiation with microwaves.

An exemplary system for carrying out the present invention is shownschematically in FIG. 1. The system comprises a microwave oven 1 havinga microwave generator 12. A coolant vessel 2 is disposed in the interiorof the oven and filled with an anti-freezing coolant 21. The tissuecontainer 3 containing a treating solution 31 and a tissue 4 to betreated is centrally placed in the coolant vessel 2. The coolant vessel2 and the tissue container 3 are made of a microwave-transmittingmaterial such a glass, polypropylene or polystyrene. As describedearlier, the microwave will heat the treating solution 31 and thus thetissue 4 to irreversibly denature the tissue matrix by heat.Consequently, a cooling apparatus 7 is disposed externally to the oven 1and the coolant 31 is circulated between the coolant vessel 2 and thecooling apparatus through the associated conduit means 5 and 6. Thecoolant 21 warmed in the vessel 2 is conveyed to a heat exchanger 72 ofthe cooling apparatus through the conduit 6 and the coolant cooled thereis returned to the vessel 2 through the conduit 5 using a pump 71. Thetemperature of the coolant in the vessel 21 is monitored by a sensor 11to generate a control signal. A controller 13 controls the operationaltime of the microwave generator 12 intermittently and automatically inresponse to the control signal to maintain the temperature at a constantlevel between 0° C. and 40° C., for example, at 10° C. Means foruniformly propagating the microwaves such as a fan or the like may beprovided in the microwave oven.

The method of the present invention may be used in conjunction with aknown decellularizing method. For example, the tissue may be pre-treatedwith a detergent solution or an enzyme solution to remove the cellularcomponents and then irradiated with microwave to remove residualchemicals from the tissue by washing.

The method of the present invention finds use in the followingtreatments.

1. Soft Mammalian Tissues for Use in Transplantation

Decellularization of soft tissues obtained from donors with death due tocerebral or heart failure or xenogeneic porcine or bovine soft tissuesfor the preparation of transplantable tissues. The efficiency of removalof cellular components by washing in the subsequent step is largelyimproved. At the same time, the immunogenecity of the tissue issubstantially diminished.

2. Hard Mammalian Tissues for Use in Transplantation

Similar to soft tissues, hard tissues such as bone, cartilage or teethmay be decellularized to prepare transplantable tissues.

3. Treatment of Other Biological Tissues for Medical Use Tissues ofanimal or plant origin may be treated for the purpose of destructingcells therein.

EXAMPLES

1. Fresh porcine hearts were purchased from a breeding farm andtransported at 4° C. The warm ischemic time of the heart was controlledwithin 20 minutes. Pulmonary valves and blood vessels were excised andwashed with Hank's solution and immersed in a 1% solution of TritonX-100. Using the system schematically shown in FIG. 1, the excisedtissues were irradiated with microwave intermittently at 20° C. toremove cells. After treating the tissue was washed with PBS to removeresidual cells. Specimens of the decellularized tissue were stained withHE and histologically evaluated by the microscopic observation.

As shown in the photograph of FIG. 2, the porcine pulmonary valveleaflet was decellularized even in deep interior portions by theirradiation with microwave in conjunction with the treatment of thedetergent solution while the treatment with the detergent solution alonefailed to achieve decellularization.

2. Porcine heart valves purchased from the same breeding farm weredecellularized by immersing in a 1% aqueous solution of Triton X-100 for24 hours. Then the decellularized heart valves were immersed in PBS andirradiated with microwave intermittently at 10° C. for 48 hours. Asshown in the graph of FIG. 3, cytotoxic Triton X-100 was removed fromthe tissue within several days by irradiation with microwaves whileabout 3 weeks were required to remove the detergent from the tissue whenthe tissue was not irradiated with microwaves.

1. A method of decellularizing mammalian tissue comprising: a) immersing the mammalian tissue in a treatment solution comprising a detergent but lacking of a fixation chemical; b) irradiating said tissue with microwaves at a frequency of 2451 MHz for a net period of at least 1 hour while maintaining the temperature of the tissue in a range between 0° C. and 40° C.; c) washing the irradiated tissue to remove residual cells, leaving a washed tissue; whereby the tissue is decellularized even in deep interior portions if determined by: d) staining a specimen from the washed tissue from c) with hematoxylin-eosin stain and histologically evaluating the specimen by microscopic observation to determine any residual cellular nuclei; e) identifying the tissue as decellularized when the specimen is marked by absence of any residual cellular nuclei even in deep interior portions.
 2. The method of claim 1 wherein said native tissue to be treated is soft tissue including vascular vessel, heart valve, heart sac, cornea, amonion and dura.
 3. The method of claim 1 wherein said native tissue to be treated is an organ or part thereof including heart, kidney, liver, pancreas, brain and part thereof.
 4. The method of claim 1 wherein said native tissue to be treated has been pre-treated to facilitate the removal of donor cells.
 5. The method of claim 1 wherein said tissue is immersed in said treating solution received in a microwave-transmitting container which, is, in turn, in heat-exchange contact with a coolant liquid received in a microwave-transmitting vessel, and wherein said tissue is irradiated with microwaves in a microwave oven while circulating said coolant liquid through a cooling apparatus provided externally of the microwave oven. 